Overview of Lux Polysaccharide Columns,

General Method Development and

Optimization Practices in Analytical and

Preparative HPLC

Prof. Bezhan Chankvetadze

Department of Physical and Analytical Chemistry

Tbilisi State University, Tbilisi, Georgia

• History of Lux® chlorinated phases

• Lux polysaccharide based CSPs

• Column screening strategies

• Elution order reversal

• Chemo- and enantio-selectivity

• New developments

• Preparative scale separations

Outline

• Polysaccharide based materials are rather universal CSPs

• These materials can be used with normal, reversed, and polar organic modes

• Applicable for both pressure and voltage-driven separations

• Extremely high enantioselectivity can be achieved using these CSPs for various

group of analytes

• A large family of polysaccharide-based CSPs are available

• Polysaccharide-based CSPs are very useful for preparative and product scale

enantioseparations

Why shall we use polysaccharide derivatives as chiral

stationary phases (CSPs) in liquid phase separation techniques?

S

C

O

O

O

10 20

10 155

20 40 680 760

+c)

b) +

0

0

a)

0

Elution time /min

αααα=112

Enantioseparation of chiral sulphoxide with

CDCPC column

Chemistry of a chiral selector

Chemistry, morphology and nature of inert carrier

Coating or immobilization technology

Pretreatment of a packing material

Column hardware

Column packing technology

What can be optimized when developing a new

chiral column?

Chlorinated Chiral Selectors

O

O

OCONH

OCONH

CH3

Cl

CH3

Cl

CH3

Cl HNOCO

Lux® Cellulose- 4

Cellulose tris (4-chloro-3 methylphenylcarbamate)

O

O

OCONH

Cl

Me

Cl

MeOCONH

Cl

HNOCOMe

Lux® Cellulose-2

Cellulose tris(3-chloro-4 methylphenylcarbamate)

O

O

OCONH

OCONH

HNOCO

Cl

Cl

CH3

CH3

Cl

CH3

Lux® Amylose-2

Amylose tris(5-chloro-2-methylphenylcarbamate)

Substitution Pattern

Spectral Properties

Chiral Recognition Ability

(or Chromatographic Performance)

Comparative Chiral Recognition of Cellulose tris (3,5-Dimethylphenylcarbamate)

(Lux® Cellulose-1/CHIRALCEL®-OD®) and Cellulose tris (3,5-Dichlorophenylcarbamate)

Eluent: Hexane/2-Propanol (90:10). Flow rate: 0.5mL/min.

CHIRALCEL, CHIRALPAK, AD, AS, OD, OD-H, and OJ are registered trademarks of DAICEL Chemical Industries, Ltd.

Intramolecular Hydrogen Bonding Between

the Adjacent Glucopyranose Units in Polysaccharide

Phenylcarbamates

23

2

3

C

Phenyl

Phenyl

CO

O

O

O

O

N H

O

O

H N

The Carbamate Moiety (-CONH-) Plays a Dual Role

in the Structure of Polysaccharide Phenylcarbamates:

1) It is most likely interaction site of the chiral analyte with the chiral

selector and thus responsible for chiral recognition ability

2) It facilitates a formation of intramolecular hydrogen bonding between

adjacent glucose unites in the structure of polysaccharide

phenylcarbamate and thus, it is responsible for its higher ordered

secondary structure and solubility in certain solvents

Designed Synthesis of Cellulose-based Chiral Stationary Phases

(Correlation between Spectroscopic Characteristics and

Chromatographic Performance)

IR Spectra in N-H range for

chloromethylphenyl-

carbamate derivatives of

cellulose (1a, 1c, 1d, 1e and 1f)

FT-IR

1H NMR spectra of cellulose phenyl

carbamate derivatives

(a) 1a, (b) 1b, (c) 1c, (d) 1d, (e) 1e

and (f) 1f

NMR

B. Chankvetadze et al. J. Chromatogr. A, 670, 1994, 39-49

1

Designed Synthesis of Cellulose-based Chiral Stationary Phases

(Correlation between Spectroscopic Characteristics and

Chromatographic Performance)

CD spectra of cellulose phenyl

carbamate derivatives (1a, 1c, 1d,

1e and 1f) in THF

CD

Dependence of Rs/N for 2,2’-

diamino-6,6’-dimethylbiphenyl on

the N-H chemical shift of cellulose

phenylcarbamate derivatives (1a, 1c,

1d, 1e, and 1f)

HPLC

B. Chankvetadze et al. J. Chromatogr. A, 670, 1994, 39-49

Review by: C. Yamamoto, Y. Okamoto Bull. Chem. Soc. Jpn., 2004, 227-257

a). The Sign in parentheses represents the optical rotation of the first-eluted enantiomer. Eluent: Hexane/2-Propanol (90:10); flow rate:

0.5mL/min. b). Eluent: Hexane/2-Propanol (98:2); 0.5mL/min.

Table 1. Separation Factors (α) in the Resolution of a-j on Cellulose and Amylose Phenylcarbamates

Amylose DerivativesCellulose DerivativesLux® Cellulose-1/CHIRALCEL® OD®

Chloro-methyl derivative (Lux Cellulose-4) is more universal

Comparative Chiral Recognition of Chlorinated and non-

Chlorinated Chiral Selectors

FT-IR and CD spectra of

tris(chloromethylphenylcarbamates) of amylose

Separation of enantiomers of cobalt

(III) acetylacetonate on (a) 1e, (b) 1b

and (c) 1d.

Separation of enantiomers of 2,2’-

dihydroxy-1,1’-binaphthyl on (a) 1e, (b)

1b and ( c ) 1d. Flow rate 1.0mL/min.

B. Chankvetadze et al. J. Chromatogr. A, 694, 1995, 101-111

Designed Synthesis of Amylose-based Chiral Stationary Phases

(Correlation between Spectroscopic Characteristics and

Chromatographic Performance)

Lux® Cellulose-1/CHIRALCEL® OD® Cellulose Derivatives

a). The Sign in parentheses represents the optical rotation of the first-eluted enantiomer. Eluent: Hexane/2-Propanol (90:10); flow rate:

0.5mL/min. b). Eluent: Hexane/2-Propanol (98:2); 0.5mL/min.

Separation Factors (α) in the Resolution of a-j on Cellulose and Amylose Phenylcarbamates

Amylose DerivativesCHIRALPAK® AD®

Lux Amylose-2

Review by: C. Yamamoto, Y. Okamoto Bull. Chem. Soc. Jpn., 2004, 227-257

Comparative Chiral Recognition of Chlorinated

and non Chlorinated Chiral Selector

CHIRALCEL, CHIRALPAK, AD, AS, OD, OD-H, and OJ are registered trademarks of DAICEL Chemical Industries, Ltd.

Chemistry of a chiral selector

Chemistry, morphology and nature of inert carrier

Coating or immobilization technology

Pretreatment of a packing material

Column hardware

Column packing technology

What can be optimized when developing a new

chiral column?

The pressure stability at least up to 300 bar

pH stability in the range of 2.0-9.0 pH units(the applications up to pH 11.0 are described in the literature)

Applicability under normal-, reversed- and polar organic mobile phase conditions

High plate number

Applicability for SFC separations

Easy conversion of analytical to prep-scale separations

Plus all common advantages of polysaccharide-based chiral columns.

Due to careful optimization of all above mentioned parameters all

chiral columns in the Lux series provide:

Portfolio Overview:

Lux® Phases Chiral Selectors

Commercial name

Polysaccharide Type of chiral selector

Substituent

Lux Cellulose-1 Cellulose Carbamate Me

Lux Cellulose-2 Cellulose Carbamate Me, Cl

Lux Cellulose-3 Cellulose Benzoate Me

Lux Cellulose-4 Cellulose Carbamate Me, Cl

Lux Amylose-2 Amylose Carbamate Me, Cl

Lux® Polysaccharide-Based CSPs

Cellulose triscarbamate Amylose triscarbamate

Lux® Cellulose-1 vs. CHIRALCEL® OD-H®

CHIRALCEL, CHIRALPAK, AD, AS, OD, OD-H, and OJ are registered trademarks of DAICEL Chemical Industries, Ltd.

Polar organic mode

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24

mv

-100

0

100

200

300

400

500

600

700

800

mv

-100

0

100

200

300

400

500

600

700

800

1: 220 nm, 4 nm

BIFONAZOLE_LUX1_1

In SFC with Bifonazole

Method Information:

Column size: 250 x 4.6 mm

Flow Rate: 2.5mL/min

15% MeOH:0.1%DEA

85% CO2

Detection: UV @ 220 nm

Temperature: 35°C

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24

mv

-100

0

100

200

300

400

500

600

700

800

900

mv

-100

0

100

200

300

400

500

600

700

800

9001: 220 nm, 4 nmBIFONAZOLE_102308_1

2

Chiralcel-OD-H

Lux-Cellulose-1

N

N

QC test Chromatogram n=5 Symmetry Efficiency

CHIRALCEL® OD-H® 0.62 15312

Lux 5u Cellulose- 1 0.74 17768

Lux shows a 17% increase in peak symmetryand a 14% increase in peak efficiency

Symmetry vs. Efficiency

Lux® Cellulose-1 vs. CHIRALCEL® OD-H®

CHIRALCEL, CHIRALPAK, AD, AS, OD, OD-H, and OJ are registered trademarks of DAICEL Chemical Industries, Ltd.

Lux® Cellulose-2 vs. CHIRALCEL® OZ-H®

CHIRALCEL OZ-H is a registered trademarks of DAICEL Chemical Industries, Ltd.

Lux 5µ Cellulose-2

Rs: 2.43

Chiralcel OZ-H

Rs: 0.92

Bupivacaine (0.1%DEA in Hexane/IPA (90:10) )

Lux 5µ Cellulose-2

Rs: 2.30

Chiralcel OZ-H

Rs: 1.84

Doxylamine (0.1%DEA in Hexane/IPA (80:20) )

Lux® Cellulose-3 vs. CHIRALCEL® OJ-H®

CHIRALCEL OJ-H is a registered trademarks of DAICEL Chemical Industries, Ltd.

Lux® Cellulose-3 vs. CHIRALCEL® OJ-H®

(Separation of stereoisomers of difenoconazole)

Hex:EtOH 90:10 V/V

Flow rate: 1 ml/min

Lux® Cellulose-3

CHIRALCEL® OJ-H®

CHIRALCEL OJ-H is a registered trademarks of DAICEL Chemical Industries, Ltd.

Coated vs. covalently immobilized polysaccharide-based

chiral columns

Coated vs. Immobilized CSPs

CHIRALPAK® AD- H® CHIRALPAK® IA™

Ghanem, L. Naim, J. Chromatogr. A 1101, 2006, 171-178

CHIRALCEL, CHIRALPAK, AD, IA are registered trademarks of DAICEL Chemical Industries, Ltd.

Ghanem, L. Naim, J. Chromatogr. A 1101, 2006, 171-178

Amylose triscarbamate

Coated vs. Immobilized CSPsLux® Cellulose-1 vs. CHIRALPAK® IB™

CHIRALPA IB is a trademarks of DAICEL Chemical Industries, Ltd.

Coated vs. Immobilized CSPsLux® Cellulose-1 vs. CHIRALPAK® IB™

Percentage of Compounds Resolved on Coated and Immobilized CSPs using Hex:IPA

CHIRALPA IB is a trademarks of DAICEL Chemical Industries, Ltd.

Method Development

•Column complementarity

•Mobile phase complementarity

•Mobile phase additives

•Temperature

•Elution order reversal

•Chemo- and enantioselectivity

Separation of Flavanone Using

Lux® Amylose-2 with NP and PO

0 10 20 30 t, min

CH3CN

Hexane/2-Propanol (9:1)

Complementary Chiral

Recognition in NP and RP

NP (Hexane/IPA)

Columns Rs

Lux Cellulose-1 0

Lux Cellulose-2 0

Lux Amylose-2 0

OH

F

O

Flurbiprofen

min0 2 4 6 8 10

mAU0

50

100

150

200

250

300

DAD1 D, Sig=220,8 Ref=off (F:\PHEN4157\HPCHEM\1\DATA\LP0209\LP206091.D)

7.585

8.510

Lux Amylose-2Rs: 3.15 (CH3CN/aq* (6:4)

min0 2 4 6 8 10

mAU0

50

100

150

200

DAD1 D, Sig=220,8 Ref=off (F:\PHEN4157\HPCHEM\1\DATA\LP0109\LP901103.D)

6.603

Lux Cellulose-2 Rs: 0 (CH3CN/aq* (6:4)

min0 2 4 6 8 10

mAU0

50

100

150

200

250

300

DAD1 D, Sig=220,8 Ref=off (F:\PHEN4157\HPCHEM\1\DATA\LP0109\LP901027.D)

6.769

Lux® Cellulose-1Rs: 0 (CH3CN/aq* (6:4)

* aq: 0.1% HAc

Separation of Bifonazole by SFC

M i n u t e s

0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4

mv

- 5 0

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

3 5 0

4 0 0

4 5 0

5 0 0

5 5 0

6 0 0

6 5 0

mv

- 5 0

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

3 5 0

4 0 0

4 5 0

5 0 0

5 5 0

6 0 0

6 5 01 : 2 2 0 n m , 4 n m

B I F O N A Z O L E _ L U X 2 _ 1 0 2 2 0 8 _ 2

MP:MeOH+0.1%DEA/CO2(15:85)

Lux 5 µm Cellulose-2

M i n u t e s

0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4

mv

- 1 0 0

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0

7 0 0

8 0 0

mv

- 1 0 0

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0

7 0 0

8 0 0

1 : 2 2 0 n m , 4 n m

B I F O N A Z O L E _ L U X 1 _ 1

MP: MeOH+0.1%DEA/CO2(15:85)

Lux® 5 µm Cellulose-1 N

N

Oct 2010-Oct 2012

244 Accepted projects

161 Successful projects

Chiral Screening Summary

244 Accepted projects

161 Successful projects

Chiral Separation Mode Statistics

244 Accepted projects

161 Successful projects

Reverse Phase Conditions

69% Success Rate

(111 successful projects)

244 Accepted projects

161 Successful projects

Normal Phase Conditions

55% Success Rate

(88 successful projects)

244 Accepted projects

161 Successful projects

Polar Organic Phase

20% Success Rate

(32 successful projects)

244 Accepted projects

161 Successful projects

Chiral Screening Summary

Method Development

Purpose:

- chemo- + enantioselectivity

- desired enantiomer elution order

Means:

- Chiral stationary phase (CSP)

- Mobile phase

- Separation temperature

Justification

Chemo- + enantioselectivity:

- resolve chiral drugs and impurities

- chiral drugs and metabolites

Desired enantiomer elution order:

- analysis

- purification

Chemo + enantioselectivity

cis-Diltiazem

Compound A

13

.29

10

.52 Lux 5µm Cellulose-1

Rs: 3.92

MP: 0.1 %DEA in Hexane/Ethanol (95:5)

Lux 5µm Amylose-2

Rs: 1.21

MP: 0.1 %DEA in Hexane/Ethanol (8:2)

Separation of Stereoisomers of cis-

Diltiazem on Lux® Chiral Columns

Unpublished result

N-desmethyl cis-

diltiazem

Compound BB

A

Lux 5µm Cellulose-2

Rs: 0.83, 0.63

MP: 0.1 % FA Hexane/Ethanol (8:2)

Lux 5µm Cellulose-1

Rs: 3.80

MP: 0.1 % FA Hexane/Ethanol (8:2)

Cyclothiazide

Lux 5µm Amylose-2

Rs: 1.22, 4.09

MP: 0.1 % FA Hexane/Ethanol (8:2)

Nadolol

Separation of Stereoisomers of Cyclothiazide

and Nadolol on Lux® Chiral Columns

Unpublished result

Unpublished result

Mobile phase: n-Hex/EtOH99/1 (v/v), 1 ml/min.

Separation of Stereoisomers

of Ethyl-3-methyl-3-phenylglycidate

and Ethyl-3-phenylglycidate on Lux® Cellulose-4

Separation of stereoisomers of difenoconazole

n-Hex/EtOH 90:10 v/v

Flow rate: 1 ml/min

Lux® Cellulose-3

2S,4R

2R,4S

2R,4R

2S,SR

F. Dong, J. Li, B. Chankvetadze, Y. Cheng, X. Liu, J. Xu, X. Chen, Y. Li, C. Bertucci,

D. Tedesco, R. Zanasi, Y. Zheng, The chiral triazole fungicide difenoconazole:

absolute stereochemistry, stereoisomer bioactivity, aquatic toxicity and environmental

behavior in vegetable and soil, Environmental Science & Technology, 47, 2013, 3386-3394.

Lux Cellulose-4 0.01% 2-propanol

Proprietary agrochemical product-1

RC

E

RT

Z

SC

ES

TE

ST

ZS

CZ

RT

E

RC

Z

Lux Cellulose-1

Enantiomer elution order

Chiral stationary phase (CSP)

Mobile phase

Separation temperature

Column Complementarity

Enantiomer Elution Order

L

DLux Cellulose-4

D

L

Lux Cellulose-3

D

L

Lux Cellulose-1

L

DLux Cellulose-2

FMOC-IsoLeucine in normal phase

300

300

150

150

30

0

10 20

0

mAU

min

Cellulose tris (3,5-dimethylphenylcarbamate)

Lux Cellulose-1

Amylose tris (3,5-dimethylphenylcarbamate)

R

S

Naproxen

Mobile phase: n-hexane/ethanol/formic acid

92/8/0.1 (v/v/v)

The effect of polysaccharide backbone on the

enantiomer recognition pattern

10 20

030

min

200

100

200

100

0

mAU

Cellulose Carbamate vs. Cellulose Benzoate

Naproxen

Mobile phase: n-hexane/ethanol/formic acid

92/8/0.1 (v/v/v)

Cellulose tris (4-methylbenzoate)

Lux Cellulose-3

5 10

0

20

min15

200

100

200

100

0

mAU

The effect of substituent position

n-hexane/2-propanol/formic acid

(99.5/0.5/0.1, v/v/v)

Ibuprofen

Enantiomer elution order

Chiral stationary phase (CSP)

Mobile phase

Separation temperature

(-)

(+)

(+)

(+)(-)

(-)

b

c

a

Analyte: Bifonazole

Column: Lux Amylose-2

Methanol

Ethanol

Acetonitrile

The Effect of Mobile Phase on

Enantiomer Elution Order

The effect of a type of

alcohol/polar modifier

200

100

300

150

0

mAU

10 20

030

min

n-hexane/2-propanol/formic acid (95/5/0.1, v/v/v)

n-hexane/ethanol/formic acid (95/5/0.1, v/v/v)

Chiral column: Lux Amylose-2

n-Hex/IPA/FA=60/40/0.1

n-Hex/IPA/FA=85/15/0.1

D

L

D,L

L

D

n-Hex/IPA/FA=95/5/0.1

The effect of IPA concentration on

enantiomer elution order in HPLC

Lux Cellulose-1FMOC-

isoleucine

0.1 %FA in Hexane/IPA(85:15)D

L

0.3 %FA in Hexane/IPA(85:15)

0.5 %FA in Hexane/IPA(85:15)

1.0 %FA in Hexane/IPA(85:15)

The Effect of FA Concentration

on Enantiomer Elution Order in HPLC

D,L

L

D

LD

CH3

OHO

CH3

H O

H

C

N

O

Enantioseparation

of Fmoc-(D,L)-Isoleucine

*

L. Chankvetadze et al. Journal of Chromatography A, 2011, 6554-6560

K.S.S. Dossou et al. Journal of Separation Science, 34 (15), 2011, 1772-1780

Enantiomeric Impurity

Determination in S-Amlodipine

0 5 10 15 20 25 30 35 40

0

50

100

150

200

250

mA

U

Time (min)

S

S

R

R

ACN/0.1% DEA/0.02% FA

0 5 10 15 20

0

50

100

150

200

250

mA

U

Time (min)

R

S

S

R

ACN/0.1% DEA/0.01% FA

The Effect of Addition of Acidic Additive

to Basic Mobile Phase on the Elution Order of Amlodipine

R

S

R

S

R

S

Time (min.)

ACN/H2O/DEA/FA (95:5:0.1:0.04)

ACN/H2O/DEA (95:5:0.1)

ACN/H2O/DEA/FA (95:5:0.1:0.06)

The Reversal of Elution Order

COOCH2CH3

CH2OCH2CH2NH2H3C

H3COOC

ACN/H2O/DEA/FA (90:10:0.1:0.03)

ACN/H2O/DEA/FA (90:10:0.1:0.3)

S

R

ACN/H2O/DEA (90:10:0.1)

S

R

R+S

No Reversal of Elution Order

COOCH2CH3

CH2OCH2CH2NH2H3C

H3COOC

Aqueous mobile phase:

Does this always mean

a reversed-phase behaviour?

R

R

R

R

R

R

R

R

S

S

S

S

S

S

S

S

ACN/DEA = 100/0.1

ACN/H2O/DEA = 90/10/0.1

ACN/H2O/DEA = 80/20/0.1

ACN/H2O/DEA = 70/30/0.1

ACN/H2O/DEA = 60/40/0.1

ACN/H2O/DEA = 50/50/0.1

ACN/H2O/DEA = 40/60/0.1

ACN/H2O/DEA = 95/5/0.1

t, min

Aqueous mobile phase:

Does this always mean

a reversed-phase behaviour?

Amlodipine

Column: Lux Cellulose-4

Effects observed:

Decrease of enantioselectivity

Disappearance of enantioselectivity

Increase of enantioselectivity

Appearance of enantioselectivity

Reversal of enantiomer recognition pattern

Effects observed:

Decrease of enantioselectivity

Disappearance of enantioselectivity

Increase of enantioselectivity

Appearance of enantioselectivity

Reversal of enantiomer recognition pattern

ACN/DEA=100/0.1

ACN/DEA/FA=100/0.1/0.1

R

S

R

S

0

20

40

0

20

40

20 40

20 40mAU

mAU

min

min

Carvedilol

Lux Cellulose-1

Decrease of enantioselectivity

RS

R

S

0

20

40

60

0

20

40

10 20

10 20mAU

mAU

min

min

IPA/DEA=100/0.1

IPA/DEA/FA=100/0.1/0.1

Amylose tris(3,5-dimethylphenylcarbamate)

Effects observed:

Decrease of enantioselectivity

Disappearance of enantioselectivity

Increase of enantioselectivity

Appearance of enantioselectivity

Reversal of enantiomer recognition pattern

a)

b)

0

40

80

120

0

200

400

600

10

10

20

20

R+S

R

S

mAU

mAU

ACN/DEA=100/0.1

ACN/DEA/FA=100/0.1/0.1

min

min

Lux Cellulose-2

Disappearance of enantioselectivity

Pindolol

Effects observed:

Decrease of enantioselectivity

Disappearance of enantioselectivity

Increase of enantioselectivity

Appearance of enantioselectivity

Reversal of enantiomer recognition pattern

IPA/DEA=100/0.1

IPA/DEA/FA=100/0.1/0.1

R

S

R

S

5 15 25

5 15 25

40

80

0

0

20

40

mAU

mAU

min

min

35

35

The effect of acidic additive on separation of basic compound

Propranolol

Column: Lux-Cellulose-1

R

S

R

S

20 40

20 40

0

40

80

40

80

mAU

mAU min

min

IPA/DEA=100/0.1

IPA/DEA/FA=100/0.1/0.1

The effect of acidic additive on separation of basic compound

Column: Lux-Cellulose-1

Pindolol

ACN=100%

ACN/DEA=100/0.1

ACN/ACA=100/0.1

R

S

R

S

R

S

0

100

200

50

100

0

0

100

200

10 20

10 20

10 20

mAU

mAU

mAU

min

min

min

Lux Amylose-2

Increase of enantioselectivity

Pindolol

Effects observed:

Decrease of enantioselectivity

Disappearance of enantioselectivity

Increase of enantioselectivity

Appearance of enantioselectivity

Reversal of enantiomer recognition pattern

R+S

RS

IPA/DEA=100/0.1

IPA/DEA/FA=100/0.1/0.1

10 20

10 20

0

100

200

0

200

400

600

mAU

mAUmin

min

The effect of acidic additive on separation of basic compound

Propranolol

Column: Lux-Cellulose-4

ACN/DEA=100/0.1

ACN/DEA/FA=100/0.1/0.1

0

0

40

80

50

100

150

10 20 30

10 20 30

mAU

mAUmin

min

Carazolol

Lux Cellulose-4

Appearance of Enantioselectivity

R+S

RS

RS

ACN/DEA=100/0.1

ACN/FA=100/0.1

ACN/DEA/FA=100/0.1/0.1

10 20

10

10

20

20

0

0

0

50

100

50

100

200

400

mAU

mAU

mAU

min

min

min

The effect of acidic additive on

separation of basic compound

Propranolol

Column: Lux-Amylose-2

ACN=100%

ACN/DEA=100/0.1

ACN/ACA=100/0.1

R+S

R+S

RS

10 20 30

10 20 30

10 20 30

0

50

100

0

20

40

60

0

100200

300

mAU

mAU

mAU

min

min

min

Propranolol

Lux Cellulose-1

Appearance of Enantioselectivity

Effects observed:

Decrease of enantioselectivity

Disappearance of enantioselectivity

Increase of enantioselectivity

Appearance of enantioselectivity

Reversal of enantiomer recognition pattern

R

S

R

S

0

100

200

300

0

50

100

150

5 15 25

5 15 25

mAU

mAU

IPA/DEA=100/0.1

IPA/DEA/FA=100/0.1/0.1

min

min

Lux Cellulose-4

Carvedilol

Reversal of Enantiomer Elution Order

ACN/DEA=100/0.1

ACN/FA=100/0.1

ACN/DEA/FA=100/0.1/0.1

R

S

R

S

S

R

0

20

40

040

80

120

2000

400600

a)

b)

c)

mAU

mAU

mAU

min

min

min10 20 30

10 20 30

10 20 30

The effect of acidic additive on

separation of basic compound

Carvedilol

Column: Lux Cellulose-4

R

S

R

S

0

100

200

300

0

100

200

300

400

IPA/DEA=100/0.1

IPA/DEA/FA=100/0.1/0.1

10

10 20

20

mAU

mAU

min

min

Reversal of Enantiomer Elution Order

Lux Cellulose-4

Pindolol

R

S

S

R

0

10

20

30

0

40

80

10 20

10 20

ACN/DEA=100/0.1

ACN/DEA/FA=100/0.1/0.1

mAU

mAU

min

min

Reversal of Enantiomer Elution Order

Carvedilol

Lux Cellulose-4

1S,2S

1R,2R

1R,2R

1S,2S

ACN/DEA=100/0.1

ACN/DEA/FA=100/0.1/0.1

0

20

40

40

0

80

10 20

10 20

mAU

mAU

min

min

Reversal of Enantiomer Elution Order

cis-Tramadol

Lux-Cellulose-4

2-Propanol=100%

2-Propanol/DEA=100/0.1

2-Propanol/FA=100/0.1

IPA/DEA/FA=100/0.1/0.1

040

80

120

050

100

150

0

100

200

0

100

200

300

10 20 30

10 20 30

10 20 30

10 20 30

R+S

R

S

S

R

SR

mAU

mAU

mAU

mAU

min

min

min

min

Reversal of Enantiomer Elution Order

Carvedilol

Lux-Cellulose-2

Enantiomer elution order

Chiral stationary phase (CSP)

Mobile phase

Separation temperature

The Effect of Temperature on

Enantiomer Elution Order

5°C

20°C

50°C

D

L

D,L

L

D

Analyte: FMOC-Isoleucine

Column: Lux Cellulose-1

Mobile phase: Hex/IPA/FA 90/10/0.1 (v/v/v)

10 20

030

800

400

800

400

800

400

mAU

5°C

55°C

75°C

R

R+S

R

S

S

t, min

Analyte: Ketoprofen

Column: Lux Cellulose-2

Mobile phase: nHex/IPA/FA 95/5/0.1 (V/V/V)

The Effect of Temperature on

Enantiomer Elution Order

(+)

(-)

(+)

(+)

(-)

(-)

(±)

55 °C

65 °C

35 °C

75 °C

Analyte: Bifonazole

Column: Lux Amylose-2

Mobile phase: Acetonitrile

The Effect of Temperature on

Enantiomer Elution Order

10 200

30

50

25

50

25

50

25

mAU R+S

S

R

0°C

25°C

75°C

S

R

t, min

Analyte: Ibuprofen

Column: Lux Cellulose-4

Mobile phase: n-Hex/IPA/FA 99.5/0.5/0.1 (v/v/v)

The Effect of Temperature on

Enantiomer Elution Order

Preparative Scale Separations

Loadability of Chiral

Stationary Phases

0.1

0.5

1.0

5.0

10

50

mg/g CSP100 Protein-based CSPs

Vancomycin CSP

Cyclodextrin-based CSPs

Tetraamide CSPs (Kromasil

Polyacrylamide (Chiraspher)

Brush-type CSPs (Pirkle)

Polysacchiride-based CSPs

Historical Preparative Chiral

Column Performance

Performance decreases as column diameter increases

• Chiral Columns do not perform as well as achiral columns

– Analytical columns offer always higher performance

• High efficiency

• Good peak symmetry

– Preparative columns have lower performance

• Lower efficiency for same particle size

• Peak tailing is prevalent

Limitations of Conventional

Slurry Packing

High pressure solvent forces

sedimentation of the slurry

After sedimentation, column is disassembled from slurry chamber and capped (as quickly as possible)

During disassembly the

bed “relaxes” and

extrudes from column

• Packed bed disturbed

• Packing density reduced

• Non-uniform density

- Lower density at the “uncapped” end

• Inherent in all slurry packed columns

• Revolution in preparative column design and

manufacturing – R&D 100 Award in 2006

• Patented Hydraulic Piston Compression

technology, adapting Dynamic Axial Compression

to high-throughput disposable column

• Removes bed collapse/voiding as a source of

column failure and greatly improved performance

• Packing is 100% micro-processor controlled

leading to vast improvements in column-to-

column consistency and overall process control

Axia™ Patented Technology

AXIA is a trademark of Phenomenex.Axia is patented by Phenomenex. U.S. Patent No. 7,674,383

Axia™ Patented Technology

Integrated axial compression technology into pre-packed preparative columns

http://www.phenomenex.com/Info/WebDocumentServe/demo9.swf

Axia™-packed Lux® Cellulose-1 250 x 21.2 mm

Media Asymmetry Plates/Meter

Lux Cellulose-1 1.37 77435

High efficiencies are maintained when scaling from analytical to

preparative dimensions

up to 50mm IDs

Column Performance

N = 70,664

αααα = 1.19

SN 461603-13

N = 75,156

αααα = 1.19

SN 456633-1Analytes: TSO

Column: Lux® Cellulose-2

Mobile Phase: Hexane:IPA 9:1 (V/V)

Ab

so

rba

nc

eA

bs

orb

an

ce

0

0 Minutes 18

Column 250 x 50 mm

Column 250 x 4.6 mm

Columns tested:

• Lux® Cellulose-1 250 x 4.6 mm, 5 µm

• Lux Cellulose-1 250 x 4.6 mm, 10 µm

• Lux Cellulose-1 250 x 4.6 mm, 20 µm

Analytical testing conditions:

• Mobile Phase: 60:40 Hex:IPA with 0.1% formic acid

• Method Type: Isocratic

• Flow Rate: 1mL/min

• Concentration of sample: ~0.5mg/mL

• Injection Volume: 10 uL

• Temperature: Ambient

• Detection: UV at 254nm

• Runtime: 15 minutes

Chiral Separation of Warfarin

2 4 6 8 10 12

mAU0

5

10

15

20

25

30

35

40

DAD1 B, Sig=254,4 Ref=off (C:\CHEM32\...IRAL PS 120831A 2012-08-31 14-59-46\WARFARIN LUX1-20U_1MG_ML_2.D)

4.6

84

7.4

60

Part α Rs

5 µm 2.4 8.7

10 µm 2.5 4.6

20 µm 2.4 2.2

10 µm

5 µm

20 µm

Chiral Separation of Warfarin

Lux® Cellulose-1

Columns tested:

• Lux® Cellulose-1 250 x 4.6 mm, 5 µm

• Lux Cellulose-1 250 x 4.6 mm, 10 µm

• Lux Cellulose-1 250 x 4.6 mm, 20 µm

Analytical testing conditions:

• Mobile Phase: 60:40 n-Hexane:Isopropanol with 0.1% formic acid

• Method Type: Isocratic

• Flow Rate: 1mL/min

• Concentration of sample: ~0.5mg/mL

• Injection Volume: 10 uL

• Temperature: Ambient

• Detection: UV at 220nm

• Runtime: 15 minutes

Chiral Separation of Lansoprazole

Part α Rs

5 µm 1.3 2.3

10 µm 1.4 2.1

20 µm 1.4 0.9

min2 4 6 8 10 12 14

mAU-50

0

50

100

150

200

DAD1 A, Sig=220,4 Ref=off (C:\CHEM32\...24 2012-08-28 14-52-09\LANSOPRAZOLE LUX1-5U 60_40 HEXANE_IPA_1.D)

2.9

31

6.3

89

7.4

47

Chiral Separation of Lansoprazole

10 µm

5 µm

20 µm

Lux® Cellulose-1

Summary for Lux® Cellulose-1

• Selectivity (α): Constant across all

particle sizes (5 µm, 10 µm, 20 µm)

• Efficiency (N): As particle size increases,

efficiency of column decreases.

• Resolution (Rs): As particle sizes

increases, resolution of peaks

decreases.

Part α N Rs

5 µm 2.4 103472 8.7

10 µm 2.5 31000 4.6

20 µm 2.4 9660 2.2

Warfarin

Part α N Rs

5 µm 1.3 66208 2.3

10 µm 1.4 25212 2.1

20 µm 1.4 7024 0.9

Lansoprazole

Columns tested:

• Lux® Cellulose-1 250x4.6 mm, 10 µm

• Lux Cellulose-1 250x4.6 mm, 20 µm

Preparative testing conditions:

• Mobile Phase: 60:40 Hex:IPA with 0.1% formic acid

• Method Type: Isocratic

• Flow Rate: 1mL/min

• Concentration of sample: 20mg/mL

• Temperature: Ambient

• Detection: UV at 254nm

• Runtime: 15 minutes

Loadability Experiments

with Warfarin

2 4 6 8 10 12

mAU0

500

1000

1500

2000

2500

3000

DAD1 B, Sig=254,4 Ref=off (C:\CHEM32\...T TEST\CHIRAL PS 120904A 2012-09-04 17-50-03\WARFARIN 10U_6MG_.D)

2 4 6 8 10 12

mAU0

500

1000

1500

2000

2500

3000

3500

DAD1 B, Sig=254,4 Ref=off (C:\CHEM32\...T TEST\CHIRAL PS 120904A 2012-09-04 16-36-45\WARFARIN 20U_6MG_.D)

Loadability 10 µm vs. 20 µm

Part α Rs

10 µm 2 1.1

20 µm 1.9 0.6

“touching bands”

10 µm

300 µL injection: 6mg of compound at 0.24% specific load

20 µm

2 4 6 8 10 12

mAU0

500

1000

1500

2000

2500

3000

DAD1 B, Sig=254,4 Ref=off (C:\CHEM32\...TEST2\CHIRAL PS 120905A 2012-09-05 14-20-08\WARFARIN 10U_14MG_.D)

2 4 6 8 10 12

mAU0

500

1000

1500

2000

2500

DAD1 B, Sig=254,4 Ref=off (C:\CHEM32\...TEST2\CHIRAL PS 120905A 2012-09-06 11-10-52\WARFARIN 20U_14MG_.D)

“Touching bands”

Part α Rs

10 µm 2.1 0.6

20 µm <1.1 0

700 µL injection: 14 mg of compound at 0.55% specific load

Loadability 10 µm vs. 20 µm

No resolution

10 µm

20 µm

Axia™ Column Family

Axia Packing Process now scaled to provide three diameters

and three lengths for Lux® products

21.2 mm

50 mm

30 mm

New Developments

Enantioseparation with Chromolith Si (R) modified by coating

of polysaccharide derivative on the surface of silica monoliths

Column size: 50 x 4.6 mm

Mobile phase: n-Hex/2-Propanol=9/1

Flow rate: 20 ml/min

Backpressure: 163 bar

t1=12.8 sec

t2 =23.0 secC CH

2OH

CF3

H

CH

OH

CF3

0 2 4 t, min.

a)

CH C

OOH

t, min.0 2 4

CH3CH3

OH OH

t, min.0 2 4

b) c)

(+)

(-)

(+)

(-) (-)

(+)

Enantioseparation of some chiral chemicals on the

monolithic capillary column coated with cellulose

tris(3,5-dimethylphenylcarbamate)

Capillary dimension: 100 mm x 15 cm; Mobile phase: n-Hex/2-Prop=9/1

CH

OH

CF3

0 15 30 45 t, sec.

(-)

(+)

Fast LC enantioseparation using monolithic capillary column

modified with cellulose tris(3,5-dimethylphenylcarbamate)

Capillary dimension: 100 mm x 15 cm

Mobile phase: n-Hex/2-Prop=9/1

t1~9 sec

t2~22 sec

Fused-core silica based CSPs

Enantioselective peak focusing in HPLC

N=

13

44

0

N=

14

67

5

N=

17

44

5

N=

53

80

5

Run 1

Run 3

Run 7

N=

16

67

0

N=

16

36

0

N=

19

23

4

N=

51

33

8N

=5

15

63

N=

18

81

6

N=

16

43

5

N=

15

52

2

Enantioselective peak focusing

Column: Amylose

tris (3,5-dimethylphenylcarbamate)

Sample diluted with/dissolved in:

n-hexane/etanol 95/5 (v/v)

Mobile Phase:

n-hexane/etanol 99.9/0.1 (v/v)

N=

12

06

8

N=

11

86

7N

=1

71

70

N=

81

66

6

Column: Lux Cellulose-3

Sample diluted with/dissolved in:

n-hexane/etanol 95/5 (v/v)

Mobile Phase:

n-hexane/etanol 99.9/0.1 (v/v)

I would like to thank for a fruitful co-operation over several years

my colleagues from Phenomenex Inc., especially:

Dr. Tivadar Farkas

Dr. Ismail Rustamov

as well as my co-workers and students at Tbilisi State University

(Tbilisi, Georgia).

Acknowledgments